Despite significant advances in pig-to-primate organ xenotransplantation, long-term graft survival is currently limited by the development of a thrombotic microangiopathy and platelet sequestration in the grafted organ and/or a consumptive coagulopathy in the recipient. The present proposal tests homogeneous heparin sulfates (HSs) synthesized using computational modeling and enzymatic/chemical tools as outlined in Projects I (PL-I. Dr. Balagurunathan) and II (Pl-ll. Dr. Desai). We hypothesize that chemically-synthesized HSs designed to preferentially inhibit Factor Xa (FXa) and/or thrombin will ameliorate the thrombotic microangiopathy and/or consumptive coagulopathy seen in the context of pig-to-primate xenografts by inhibiting thrombin formation and platelet activation. In addition, HSs optimized to bind chemokines (from Project III) may act as chemokine decoy factors to prevent cell sequestration synergistically with anticoagulation. These HSs will be evaluated in the context of various xenotransplantation models using cells and organs from pigs genetically modified to protect against antibody/complement-mediated, inflammatory, and coagulation rejection mechanisms. These HS-based therapies will be tested by. - (i) in vitro assays to determine the best HS candidate drugs for further studies, (ii) ex vivo pig lung hemoperfusion using human blood, (iii) pig artery Tx in baboons, and (iv) pig kidney Tx in baboons. Model (ii) constitutes a short-duration model (with graft function measured in hours) allowing rapid screening of the HSs being tested, whereas models (iii) and (iv) constitute longer-duration models (days or weeks). Furthermore, the effect of the HS will be monitored (iii) in a low-antigen load model without immunosuppressive therapy, or (iv) in a high-antigen load model in which graft recipients will receive immunosuppressive therapy aimed at inhibiting the adaptive T cell response. The prime aim of the study is to identify one (or more) HS that is more specific and more efficient at inhibiting the development of thrombotic microangiopathy and consumptive coagulopathy than is the current clinically-used heparin, and that, furthermore, is not associated with the side-effects of heparin, e.g., bleeding, that limit the dosage that can be administered. The second aim is to investigate the synergy between the HS identified as being optimal with the genetic manipulations in the organ-source pig, e.g., expression of human thrombomodulin directed towards inhibiting thrombotic microangiopathy and consumptive coagulopathy. RELEVANCE (See instructions): The transplantation of organs from genetically-engineered pigs would provide an alternative source to human organs for clinical transplantation, but is currently complicated by coagulation disturbances that can be fatal. The administration of specific synthetic heparan sulfates that will be designed and investigated in this proposal are likely to prevent these coagulation disturbances, thus enabling xenotransplantation to advance to clinical trials.